Approximately 23.6 million Americans (7.8% of the population) have diabetes - most needing to produce more insulin because of insulin resistance, yet nearly all with pancreatic -cell dysfunction. In -cells, insulin synthesis begins with the precursor, preproinsulin, which must undergo co-translational translocation into the endoplasmic reticulum (ER), signal peptide (SP) cleavage, and downstream proinsulin folding. These earliest events are critical to insulin biosynthesis, but they are relatively understudied. Over the past three years, four preproinsulin SP mutations have been reported to cause human diabetes that make investigation of these earliest events especially timely. While it is known that insulin haploinsufficiency does not cause diabetes, patients with preproinsulin SP mutations are heterozygotes, suggesting that mutants act in a dominant- negative fashion. The molecular mechanisms of -cell failure caused by these mutants remain unknown. Interestingly, diabetes phenotypes associated with the SP mutants ranges from severe neonatal-onset diabetes caused by A(SP24)D, to mild adult-onset diabetes associated with R(SP6)C or H. I hypothesize that these two classes of SP mutants cause -cell failure through two distinct mechanisms. In one case, I propose that inefficient co-translational translocation [of R(SP6)C] causes cytosolic accumulation of untranslocated mutant that is slowly toxic to -cells, leading to adult-onset diabetes that may be akin to the pathogenesis of Alzheimer's and some other neurodegenerative diseases. In the other case, I propose that failed SP cleavage [of A(SP24)D] disturbs downstream proinsulin folding, causing ER retention of the mutant that abnormally interacts with co-expressed wild-type (WT) proinsulin and blocks its ER exit in trans, decreasing insulin production and initiating severe insulin-deficient diabetes in early life. This proposal aims to better understand the coordination of the earliest events of insulin biosynthesis and define the molecular mechanisms of -cell failure caused by defects of those events. The ultimate goal is to develop novel strategies to prevent development of diabetes caused by misfolded (pre)proinsulin. Three Specific Aims are proposed: 1) To examine coordination of preproinsulin co-translational translocation, SP cleavage, and downstream proinsulin folding; 2) To define genetic manipulations that would allow WT proinsulin to escape from blockade caused by mutant (pre)proinsulins; 3) To identify small molecules that could prevent -cell failure caused by mutant preproinsulins. Accumulating evidence suggests that ER stress and proinsulin misfolding plays a role in the pathogenesis of the most common form of diabetes (type 2) which does not involve any preproinsulin coding sequence mutations. These new diabetogenic mutants, in which underlying (pre)proinsulin mishandling-misfolding is unequivocal, are ideal models for understanding molecular mechanisms of -cell failure, and for testing experimental therapies aiming at preventing diabetes.